Fast Desalting of Proteins Using a Novel High Mechanical Strength Gel Filtration Column

Presider(s):

Start Time:

( Slot # 5 )

Date:

Monday, March 12th, 2012

Location:

Red Area on Exposition Floor, Aisles 1300-1500

Keywords:

HPLC Columns, Liquid Chromatography, Pharmaceutical, Protein

Co-Authors

Name

Affiliation

Eksteen, Roy

Tosoh Bioscience LLC

Abstract Content

Desalting by Gel Filtration Chromatography (GFC) is the preferred method in biochemical laboratories to reduce the salt concentration or exchange the buffer of a biopolymer solution, with speed being the main advantage of GFC over dialysis. Requirements for a fast desalting column are (1) an inert matrix, (2) a large pore volume that is fully accessible to common salts and buffer components, (3) a pore size distribution that excludes the component(s) of interest from accessing the pores, and (4) sufficient mechanical strength to allow the use of the column in standard HPLC equipment. While dextran, cellulose and polyacrylamide are commonly used as porous gel matrices in desalting columns, such packing materials can withstand only minimal pressure. A recent report stated that the mechanical strength of polyacrylamide gel could be increased by a continued cross-linking process (1). The increase in particle strength combined with a very narrow particle size distribution allowed the use of a particle size of 15m. The exclusion limit of the packing material (~2500 Da) was determined with polyethylene glycol standards. Here we report on the application of TSKgel BioAssist DS columns for fast desalting of protein and polynucleotide samples at analytical and semi-preparative scale. Our results demonstrate that buffer and salt concentrations of protein solutions can be reduced at least 50-fold within 5 minutes, with minimal dilution of the protein concentration and excellent recoveries down to ng protein injected.(1) US Patent 7659348 B2, February 9, 2010